tbuckley/cabot-search huggingface dataset:
Abstract P2170: Rescue Of Ctni Null Mice By Application Of Aav-9 Encoding Ctni Gene
Authors: Dustin Gerber, John Gacitua, Andrue Hackshaw, Howard M Prentice, Xupei Huang
Journal: Circulation Research (2023)

Background: Heart diastolic dysfunction can be caused by cardiac troponin I (cTnI) deficiencies or mutations. Our lab has created a unique cTnI gene knockout mouse model, demonstrating that homozygous cTnI null mice (-/-) die at postnatal day 18 (p18). In this study, we have applied gene therapy using adeno-associated virus serotype 9 (AAV9) carrying wild-type cTnI gene to rescue mice from a lethal cTnI knockout phenotype. Methods: The proposed gene therapy strategies involve novel approaches delivering AAV9 via intraperitoneal injection in newborn cTnI mice (-/-), employing a cardiac-specific promoter (chicken cTnT promoter) to express cTnI protein in myocardial cells. Results: Mice treated with doses ranging from 2e10 10 vg/g (vector genome per gram) to 8e10 10 vg/g at p10 displayed an increased life span compared to mice without treatment. 5e10 10 vg/g multi-injection two-dose treatment at p10 and p14 demonstrated 22 additional days of life past p18 ( Figure A) . Using echocardiography, treated mice showed significant improvement in left ventricular diastolic function compared to mice without treatment. In addition, western blotting of cardiac samples demonstrated restored cTnI proteins in treated mice. Conclusion: The in vivo gene therapy approach shows that our mouse model can survive 2-3 weeks longer than untreated cTnI null mice. These data suggest AAV-9 serotype with a cardiac-specific promoter is a promising tool in treating cardiomyopathies by restoring cardiac proteins and reversing diastolic dysfunction.
Abstract P2177: Autophagy-based Drugs Can Be Used To Treat Anthracycline-induced Cardiotoxicity
Authors: Song Zhang, Johanna E Jacobs, David Mondaca-Ruff, Xiaolei Xu, Yonghe Ding, Joerg Herrmann
Journal: Circulation Research (2023)

Background: Anthracyclines are highly effective chemotherapeutics, but their use is limited by Anthracycline-Induced Cardiotoxicity (AIC) with notable decline in ejection fraction (EF). Recovery of cardiac function is slow and often only partial, mandating novel and more efficacious treatment approaches to AIC. Hypothesis: Based on high throughput genetic screening in zebrafish we identified autophagy activation, e.g. via Atg7, a key protein in the induction of autophagy, as a potential novel translational therapeutic approach to AIC. Methods: In a mouse model of cardiotoxicity (4 x weekly 5 mg/kg intravenous doxorubicin treatment) two approaches were tested starting one week after the last treatment: 1) AdenoAssociated Viral vector 9-mediated overexpression of Atg7 in the myocardium (AAV9-ATG7, one time injection) and 2) 3 FDA approved autophagy activating (FAA) drugs (pravastatin (40 mg/kg), spironolactone (20 mg/kg) and empagliflozin (10 mg/kg), by daily gavage). Echocardiograms were performed at baseline, before start of therapy and after 28 days before sacrifice. Results: All three FAA drugs were able to rescue AIC and Atg7 overexpression by AAV9 alone sufficed to rescue from AIC (Figure 1). All three FAA drugs were able to either restore Atg7 levels to baseline (pravastatin and empagliflozin) or 2.5 times above baseline levels (spironolactone) versus more than 60fold above baseline by an AAV9-based approach. Conclusion: Atg7 overexpression and autophagy activation suffices to rescue from AIC and can be recapitulated to different levels by pravastatin, empaglifozin and spironolactone.
Abstract P1088: Biophysical Crowding Of <i>In Vitro</i> Culture Media Alters The Growth Rate And Collagen Production Of Cells Used For Seeding Tissue-Engineered Heart Valves
Authors: Kathryn G Henshaw, Angelina J Joyce, Renata C Freitas, John E Mayer
Journal: Circulation Research (2023)

Introduction: Ideal tissue-engineered heart valves (TEHVs) for valve replacement in congenital heart disease patients would have durability and growth potential. Endothelial colony-forming cells (ECFCs) isolated from peripheral blood are an attractive source for pre-cellularizing TEHVs with a patient’s own cells, as they are isolated non-invasively and can transform into valve endothelial and interstitial phenotypes. ECFC limitations include slow proliferation and uncertain mechanisms to control extracellular matrix (ECM) production. In other cell types, culture media “crowded” with large macromolecules limits paracrine diffusion and augments both ECM production and cell growth. Hypothesis: As a novel study of biophysical crowding on ECFCs, we hypothesized that media crowded with macromolecules dextran sulfate or Ficoll-400 would enhance the growth rate and collagen production of ovine ECFCs. Methods: ECFC lines (CD31+, α-SMA+) from n=3 animals at passages 2, 3, or 7 were cultured in media with no crowder or either Ficoll-400 or dextran sulfate at four concentrations in three separate replicates (n=10, n=120, n=384). Deposited and suspended collagen was quantified. Immunocytochemical cell scoring was quantified for vWF, VEGFR-2, CD34, vimentin, and α-SMA markers. Results and Conclusions: Anionic dextran sulfate (DS) caused significant dose-dependent increases in media soluble collagen, despite significantly lowering cell counts. Crowding with DS significantly increased CD34 positivity, a marker for anti-angiogenic and quiescent ECFC populations. Conversely, neutral Ficoll-400 significantly increased cell counts but did not alter media collagen or CD34 positivity. Both crowder groups had altered cellular morphology but did not cause reproducible differences in positivity for endothelial markers vWF and VEGFR-2 or interstitial markers vimentin and α-SMA. In conclusion, biophysical crowding of ECFC media alters cell count and morphology, CD34 positivity, and collagen production, which may allow for fine-tuning of ECFC properties when seeding onto TEHVs. Future studies will address the different effects of neutral and negatively charged crowders and how crowders impact cellular phenotype and growth on valvular scaffolds.
Abstract P3146: Cardiac Stress-induced Arrhythmias And Myocardial Infarction Via Specific Genetically Phosphodeleted Sites In Connexin 43 Channels
Authors: Manuel F Munoz, Thao Nguyen, Janet Nuno, Jorge Contreras
Journal: Circulation Research (2023)

Connexin 43 (Cx43) gap junction remodeling is observed in various cardiac pathologies and is linked with arrhythmogenesis, myocardial infarction, and sudden death. Here, we aim to characterize the contribution of Cx43 phosphorylation toward mislocalization at the intercalated disc. For this, we used two genetically phospho-deleted Cx43 knock-in mouse lines with serine residues replaced by alanine in the corresponding sites for phosphorylation via mitogen-activated protein kinase (MAPK) (S255/262/279/282A; Cx43MK4) or Ca 2+/ calmodulin protein kinase II (CaMKII) (S325A/S328A/S330A; Cx43S3A). Ca 2+ signaling, cardiac electrical signals, and arrhythmias were assessed via optical mapping and telemetry in wild type, Cx43MK4, and Cx43S3A mice. Upon basal conditions or isoproterenol stimulation, wild-type animals did not display arrhythmogenic behaviors. However, after isoproterenol treatment, Cx43S3A mice showed increased QTc and deadly arrhythmias compared to wild-type animals. Isoproterenol challenge only increased the number of single PVCs in Cx43MK4 mice when compared to wild-type. Isoproterenol also evoked myocardial infarction in Cx43S3A hearts, which is negligible in Cx43MK4 and wild-type mice. Interestingly, GAP19, a Cx43 hemichannel blocker, reduced lethal arrhythmias and myocardial infarction in Cx43S3A mice. Isoproterenol challenge induced an increase in arrhythmogenic Ca 2+ events in both Cx43S3A and Cx43MK4 hearts compared to wild-type mice. However, Cx43S3A also displayed prolonged Ca 2+ decay transients that were reduced in the presence of Gap19. Our results suggest that the absence of Cx43 phosphorylation by CaMKII leads to lateralization and opening of Cx43 hemichannels, and consequently severe arrhythmias and myocardial infarction. On the other hand, the absence of MAPK phosphorylation promotes mainly a reduction of Cx43 levels at the intercalated disc region that is only associated with mild arrhythmias.
Abstract P3152: Diabetic Upregulation Of Grk2 Impairs Adipocyte Exosome Surface Adiponectin-mediated Cardioprotection And Exacerbates Ischemic Heart Failure
Authors: Zhen Zhang, Caihong Liu, Di Zhu, Zhijun Meng, John Zhang, Wayne Lau, Bernard L Lopez, Theodore Christopher, Erhe Gao, Walter J Koch, Jianli Zhao, Xinliang (Xin) Ma, Yajing Wang
Journal: Circulation Research (2023)

Background: Type 2 diabetes mellitus (DM) significantly exacerbates ischemic heart failure (IHF) by incompletely understood mechanisms. We recently reported that DM switches adipocyte-derived exosome (Exo) from cardioprotective signaling activators in nondiabetes (ND) to vehicles carrying cytotoxic molecules from DM adipocyte to heart. However, it remains unclear how DM triggers this switch and whether blocking it can protect against DM exacerbation of IHF. Methods and Results: ND or DM (12 weeks of high-fat diet) mice were subjected to MI/R (90 minutes/4 weeks) and treated with Exo (isolated from ND epidydimal fat pads and intramyocardially injected immediately before reperfusion). Exo administration significantly attenuated IHF in ND but not in DM mice. In vitro mechanistic and in vivo concept-prove experiments were conducted to determine how Exo protects against IHF and how DM blocks this protection. In cardiomyocytes (CM) from ND mice, Exo rapidly activated multiple injury salvage kinases (ISKs, including ERK, AMPK, and ACC) and attenuated cell death, suggesting that Exo-surface molecules mediate Exo cardioprotection. Exo-Flow (a method specifically detects Exo surface molecules) experiments demonstrated that adipocyte-derived Exo carries high levels of adiponectin (APN) on their surface. APN antibody-neutralized Exo or APNKO mice-derived Exo neither activated ISKs nor attenuated IHF. Exo failed to activate ISKs and protect IHF in AdipoR1KO mice. These results demonstrated that the adipocyte Exo surface-localized APN and CM-expressed AdipoR1 are essential in protective communication between adipocyte and heart. GRK2, a kinase that phosphorylates AdipoR1 at S205 and blocks APN cardioprotection, was dramatically upregulated in DM hearts. GRK2 overexpression blocked Exo-induced ISKs activation and cellular protection in WT CM but not in AdipoR1 S205A knock-in CM. Finally, Exo administration in AAV9-mediated CM-specific AdipoR1 S205A knock-in mice significantly attenuated diabetic IHF, while it failed to protect in AdipoR1 S205E mice. Conclusions: This study sheds light on the mechanisms underlying DM exacerbation of IHF and highlights the potential of targeting the APN/AdipoR1/GRK2 axis to protect against this condition.
Abstract P1095: Studying Changes In Mitochondrial Dynamics And Energetics During Cardiac Reprogramming
Authors: Brian Spurlock, Yifang Xie, Rustem Salmenov, Jiandong Liu, Li Qian, Marazzano Colon
Journal: Circulation Research (2023)

In mammals, cardiac ischemic injury leads to loss of muscle filled in with less functional scar tissue. Direct reprogramming of the cardiac fibroblasts populating a scar to functional cardiomyocytes represents a promising potential treatment for these patients. This reprogramming involves a metabolic shift from the more glycolytic CF state to mitochondrial oxidative phosphorylation in the induced CMs, including increased mitochondrial biogenesis. Various reprogramming strategies make use of agents, which in addition to canonical roles, promote mitochondrial energetics and a dynamic shift towards mitochondrial fusion. We decided to investigate whether these mitochondrial structural and functional shifts might represent a mechanism of reprogramming. We characterized various structural and energetic properties of mitochondria in neonatal CFs, CMs, and at multiple time points during reprogramming. Using the baseline CF and CM mitochondrial data, we built a model to follow cells in pseudotime during reprogramming, and identified a time course for mitochondrial changes. We also investigated whether various mitochondrial perturbations designed to promote these changes might increase reprogramming efficiency. Promotion of mitochondrial fusion activity and biogenesis increased the proportion of reprogrammed cells. Conversely, promotion of fission and inhibitors for electron transport system enzymes inhibited reprogramming.
Abstract P3023: Endothelial Pparδ Deletion Exacerbates Endotoxemia-Induced Vascular Injury And Inflammation
Authors: Huiling Hong, Yalan Wu, Xiao-Yu Tian
Journal: Circulation Research (2023)

Rationale and Background: Impairment of vascular endothelial integrity is one of the hallmarks of acute lung injury, which underlies leukocyte infiltration and edema formation and contributes to organ dysfunction. The peroxisome proliferator-activated receptor delta (PPARδ) is a transcription factor regulating vascular endothelial cell (EC) function. Our previous study revealed that selective deletion of PPARδ in the endothelium worsens ischemic vascular injury, disrupts endothelial integrity, and exacerbates vascular inflammation in a mouse model of hindlimb ischemia. Methods and Results: In this study, we investigated whether PPARδ is protective for EC function in acute vascular injury and inflammation in the mouse lung. We performed lipopolysaccharide (LPS)-induced endotoxemia in the EC-selective PPARδ knockout mice (Ppard EC-KO ) and their wild-type littermates (Ppard EC-WT ). The mortality of Ppard EC-KO mice was higher after LPS injection. The survived Ppard EC-KO mice showed more severe lung vascular inflammation suggested by sustained leukocyte infiltration in the lung tissues and in the bronchoalveolar fluid, more pro-inflammatory cytokine production, and higher expression of EC activation markers CCL2, ICAM1, VCAM1, and E-selectin. In addition, the lung endothelium of LPS-treated Ppard EC-KO mice has degraded endothelial glycocalyx and discontinuous EC junctions, resulting in increased vascular leakage. In vitro experiments using human lung microvascular endothelial cells (HLMVECs) revealed that silencing PPARδ downregulated the expressions of ZO-1, claudin-5, and VE-cadherin, and altered their membrane distribution, leading to hyperpermeability of EC monolayer, at basal level and also in the presence of LPS or TNFα. Silencing of PPARδ also induces more activation of pro-inflammatory transcription factors including STAT1, IRF3, and IRF7. Conclusion: In summary, our study showed the protective role of endothelial PPARδ against endotoxemia-induced disruption of vascular barrier function and vascular inflammation in the lung endothelium. (Supported by Hong Kong Research Grant Council GRF 14107822 and 14105321)
Abstract P3154: Characterized Expression Of MicroRNAs In Plasma Exosome In Patients With Coronary Chronic Total Occlusion
Authors: Jon Suh, Ji-Hye Son, Inki Moon, Min Gyu Kong, Dongeon Kim, Jeong Kyu Park, Hyung Oh Choi, Hye-Sun Seo, Yoon Haeng Cho, Hun Soo Chang
Journal: Circulation Research (2023)

Backgrounds: Even with similar coronary artery occlusion, chronic total occlusion (CTO) has different clinical features from acute myocardial infarction (AMI). MicroRNAs (miRNA; miR) have been regarded as biomarker to identify heterogenous patients, as well as new therapeutic targets in cardiovascular disease. We aimed to identify exosomal miRNA important for CTO. Methods: Patients with percutaneous coronary intervention (PCI) of CTO (n=30) and AMI (n=30) were performed miRNA expression profiling via exosomal small RNA sequencing in the plasma for these groups. Receiver operating characteristic (ROC) analysis, gene ontology, and functional pathway were analyzed in the miRNA exhibiting &gt;4 absolute fold changes with FDR &lt; 0.05. Results: We detected 10 miRNAs -including miR-21-5p, miR-206, miR-215-5p, and miR-1-3p upregulated in CTO and 3 miRNAs -including miR-9-5p, miR-3529-3p, and miR-127-3p downregulated in CTO as compared to AMI (FDR q &lt; 0.05). Gene enrichment and pathway analysis showed that the target genes of the 10 upregulated miRNAs were involved in regulation of osteoblast proliferation and interleukin-4 and interleukin-13 signaling. In contrast, the target genes of the 3 down-regulated miRNAs were enriched in heart valve development and blood vessel development. We selected 7 miRNAs -including miR-21-5p, miR-206, miR-215-5p, miR-1-3p, miR-9-5p, miR-3529-3p, and miR-127-3p which were related to major pathway term in gene ontology and validated by quantitative real-time polymerase chain reaction (qRT-PCR). In qRT-PCR analysis, the expression levels of miR-9-5p and miR-127-3p of plasma exosome were both lower in CTO than those of AMI, similar to the results of exosomal small RNA sequencing. The ROC curve were analyzed in the 2 miRNAs and for the integrative miRNA expression score showed high accuracy in distinguishing between the CTO and AMI (AUC = 0.948) with 79% specificity and 97% sensitivity. Conclusions: We identified significantly different miRNA expression patterns between the CTO and AMI. The levels of these miRNAs in plasma exosomes may be promising markers that distinguish CTO from AMI.
Abstract P3039: The Biophysical Action Of Microrna-1 Is Critical To Maintaining The Homeostasis Of The Heart
Authors: Dandan Yang, Xiaoping Wan, Ege Kacira, Xianyao xu, Feng Tang, Thomas J Hund, Peter J Mohler, Isabelle Deschenes, Jidong A Fu
Journal: Circulation Research (2023)

MicroRNAs (miRs) are evolutionarily-conserved small noncoding RNAs (~22 nucleotides) and are involved in most biological events through the classical mechanism of RNA interference (RNAi). Recently, we discovered a novel action of miR to biophysically modulate the function of bound proteins. miR1, the most predominant miR in the heart, physically binds to the inward rectifier potassium channel Kir2.1, directly represses the I K1 current, and biophysically modulates cardiac electrophysiology. An arrhythmia-associated human single nucleotide polymorphism of miR1 (hSNP14A/G) specifically disrupts biophysical modulation while retaining the RNAi function. However, the physiological significance of miR’s biophysical action on the heart remains unknown. To study this, we created hSNP14A/G single nucleotide mutation on miR1-1 and miR1-2 by using CRISPR/Cas9 technology and successfully generated 14G-mutated transgenic homozygous mice (14G-Homo, miR1-1 14G/G :miR1-2 14G/G ), in which the mutated miR1 maintains the RNAi while with a specific deletion of the biophysical action. We found that 14G-Homo mice had high mortality with impaired heart pumping function and some mice developed heart failure at 6 months of age. The whole transcriptome RNA-sequencing assays revealed cardiac electrical remodeling in 14G-Homo hearts. Consistently, 14G-Homo hearts had prolonged QRS and QT intervals with slower electrical conduction through the ventricle, and 14G-Homo mice are susceptible to arrhythmia. Our patch clamping showed that 14G-Homo cardiomyocytes had significantly prolonged action potential with bigger I K1 and L-type Ca 2+ current (I Ca,L ) than WT cells. We also performed a CLIP RNA pulldown-mass spectrometry assay and identified more ion channel proteins that interact with miR1. From the validation, miR1 biophysically suppresses I Ca,L via directly binding to Cavβ2, the β subunit of the voltage-gated Ca 2+ channel. We conclude that miR1 biophysically modulates the function of the heart, which is critical to maintaining heart homeostasis. Our discovery expands the biological significance of miR biology and broadens its implication of RNA medicine development for cardiovascular diseases.
Abstract P1089: Pontin Is Necessary And Sufficient For Driving Cardiomyocyte Proliferation In Zebrafish And Mice
Authors: Hui-Min Yin, Xinlei Gao, Kaifu Chen, Caroline Burns, C. Geoffrey Burns
Journal: Circulation Research (2023)

A major barrier to human heart regeneration is the absence of cardiomyocyte (CM) cell division in the infarcted heart. Remarkably, injured hearts of adult zebrafish and neonatal mice are capable of robust heart regeneration achieved through CM proliferation. Pontin is an evolutionarily conserved member of the AAA+ ATPase superfamily of proteins that regulates transcription, chromatin remodeling, and cell cycle progression in many contexts. Nonetheless, the involvement of Pontin in regulating CM proliferation has not been assessed. Using loss- and gain-of-function models in zebrafish and mice, we have determined that Pontin is necessary and sufficient for driving CM proliferation. Zebrafish pontin mutants are devoid of CM proliferation during heart development, and CM-specific overexpression of Pontin ( pontin OE ) is sufficient to induce myocardial hyperplasia during heart development and regeneration. In mice, myocardial-specific deletion of Pontin dampens CM proliferation, leading to embryonic lethality at E12.5, while CM-specific overexpression of Pontin from P0 to P7 is sufficient to increase the CM mitotic index. To decipher the molecular mechanism by which pontin OE stimulates CM proliferation, we performed single-cell ATAC + RNA-sequencing analysis of hyperproliferative pontin OE CMs during zebrafish regeneration. We identified significant downregulation of several anti-proliferative genes, including three in the btg/tob family ( btg1 , btg2 , tob1b ). The btg2 and tob1b loci showed reduced chromatin accessibility, suggesting that they could be direct targets of Pontin’s chromatin remodeling activity. We are currently testing this hypothesis and determining if overexpression of Pontin in neonatal mice extends the regenerative window beyond P7. In conclusion, our data highlight Pontin as a novel regulator of CM proliferation, potentially by controlling the chromatin accessibility of anti-proliferative genes.